Treatment of cancer with naltrexone

ABSTRACT

The present invention provides novel therapeutic applications of low dose naltrexone (LDN). Said applications have been determined in light of the discovery by the present inventors that naltrexone acts as an antagonist of Toll-like receptor 9 (TLR9), an innate immune receptor which elicits the production of inflammatory cytokines when agonised. Chronic inflammation and TLR9 overexpression are characteristics of a number of disorders, including certain cancers. Accordingly, the present invention provides novel uses of naltrexone in the treatment of a subject having a disorder characterised by TLR9 overexpression and/or overactivity of TLR9-mediated signalling. The present invention also provides novel uses of naltrexone in the supportive care of subject having a tumour/cancer, and methods of treating and providing supportive care to a subject, comprising the administration of naltrexone.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/890,300 filed Nov. 10, 2015, which is a national phase entryunder 35 U.S.C. § 371 of International Application No. PCT/GB2014/051439filed May 12, 2014, published as WO 2014/181131, which claims priorityfrom Great Britain Patent Application No. 1308440.5, filed May 10, 2013,the disclosures of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the treatment and supportive care of a subjecthaving a disorder characterised by aberrant activity of the innateimmune system, notably certain forms of cancer.

BACKGROUND OF THE INVENTION

Naltrexone is an orally-administered opioid antagonist with the chemicalnamemorphinan-6-one,17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxy-(5a).

The molecular formula of naltrexone is C₂₀H₂₃NO₄ and its molecularweight is 341.41 in the anhydrous form (<1% maximum water content). Thechemical structure of naltrexone is shown below.

Naltrexone is commonly used as a treatment for opiate addiction. Anumber of patients, however, use low doses of naltrexone (LDN) as anoff-label treatment for a range of immune-related pathologies andcancer. There is preliminary evidence that LDN may be effective inMultiple Sclerosis (Rahn et al. 2011), Crohn's disease (Smith et al.2011) and certain cancers.

With regard to cancer, Zagon and McLaughlin (1983) and Hytrek et al.(1996) reported LDN-mediated inhibition of murine neuroblastoma andhuman colon cancer cell growth respectively, when evaluated in murinexenograft models. Furthermore, combinations of LDN with additionaltherapeutic agents have been found to be effective against the growthand progression of certain cancer types, for example Donahue et al.(2011 a) reported potent anti-proliferative effects of LDN and cisplatinon human ovarian cancer cells both in vitro and in an in vivo murinexenograft model. In the clinic, Berkson et al. (2006) described the longterm survival of a patient having pancreatic cancer with metastasis toliver, following treatment with a-lipoic acid in combination with LDN;the authors have since reported similar observations in three furtherpatients with metastatic pancreatic cancer (Berkson et al. 2009).

The role of LDN in the above observations has so far largely beenexplained by reference to the antagonistic action of LDN on the opioidgrowth factor receptor (OGFr). OGFr recognises opioid growth factor(OGF, also referred to as [Met⁵]-enkephalin), and intermittent doses ofnaltrexone appear to provide a temporary blockade that triggersupregulation of the receptor (Hytrek et al. 1996). This upregulation ofOGFr has been found to result in decreased in vitro proliferation ofcells representative of pancreatic, colorectal and squamous cellcarcinomas (Donaghue et al. 2011 b).

However, a potential mechanism for LDN as a direct immunomodulator hasbeen revealed in studies by Hutchinson et al. (2008), who demonstratedthat in addition to its effects on OGFr, naltrexone can weaklyantagonise TLR4, a member of the Toll-like receptor (TLR) family.Pattern recognition receptors such as TLRs allow immune cells to detectthe presence of pathogens or self-derived danger signals, and instructthem to trigger an immune response. Innate immune activation is anecessary step for eliciting subsequent adaptive immune responses, andgiven the powerful inflammatory potential of TLRs, their activity mustbe tightly controlled. In general, TLR stimulation elicits the inductionof feedback mechanisms, but when this fails, TLR overactivity can leadto inappropriate immune responses, immune exhaustion and/orautoimmunity.

SUMMARY OF THE INVENTION

The present inventors have found that naltrexone also acts as anantagonist of the innate immune receptor TLR9. This observation hasprofound implications for the use of LDN as an immunomodulator, and hasenabled the present inventors to determine a number of novel therapeuticapplications, notably in the field of cancer therapy and supportivecare.

According to a first aspect, the present invention provides apharmaceutical composition comprising naltrexone, for use in thetreatment of a subject having a disorder which is characterised by TLR9overexpression and/or overactivity of TLR9-mediated signalling.

According to a second aspect, the present invention provides apharmaceutical composition comprising naltrexone, for use in thesupportive care of a subject having a tumour/cancer.

According to a third aspect, the present invention provides a method oftreating a subject having a disorder which is characterised by TLR9overexpression and/or overactivity of TLR9-mediated signalling; whereinthe method comprises administering to the subject, a pharmaceuticalcomposition comprising naltrexone.

According to a fourth aspect, the present invention provides a method ofproviding supportive care to a subject having a tumour/cancer,comprising administering to the subject, a pharmaceutical compositioncomprising naltrexone.

BRIEF DESCRIPTION OF THE FIGURES

The invention is described with reference to the accompanying figures,wherein;

FIG. 1 shows antagonism of TLR9 by naltrexone. Samples and controls weretested in duplicate on recombinant HEK-293 cell lines expressing thevarious TLR and NOD receptors with an alkaline phosphatase reportergene. Cells were incubated with or without 1 μM naltrexone in anantagonist assay using the Invivogen panel of TLR and NOD signallingtransfectants. Results shown are for TLR4 and TLR9, TLR activationresults are given as optical density (O.D.) values, which have beenconfirmed in three independent experiments.

FIG. 2 shows the ability of naltrexone diluted in phosphate bufferedsaline (PBS) or H₂O to inhibit TLR9 at various concentrations, for aTLR9-transfected cell line (as described above).

DETAILED DESCRIPTION OF THE INVENTION

The discovery by the present inventors that naltrexone acts as anantagonist of TLR9 has enabled the determination of novel therapeuticapplications of LDN, notably in the field of cancer therapy andsupportive care.

Certain pattern recognition receptors, such as TLR4, are located on thecell surface, whilst others such as TLR7, TLR8 and TLR9 are found withinthe cell. As naltrexone can enter cells by passive diffusion (Cheng etal. 2009), the present inventors sought to determine whether it mightantagonise any intracellular TLR in addition to the cell-surface proteinTLR4. A striking antagonistic effect was observed for TLR9, for whichLDN inhibited recognition of CpG DNA (FIG. 1), and a subsequenttitration suggested an IC50 of approximately 50 μM (FIG. 2).Furthermore, the mild antagonistic effect on TLR4 observed by Hutchinsonet al. (2008) was replicated.

TLR9 is an innate immune receptor which, upon detection of unmethylatedCpG DNA, elicits the production of type 1 interferons and otherinflammatory cytokines (TNFa, IL-2, IL-6). The nature of the responsegenerated following TLR9 activity can vary according to theintracellular location where the receptor interacts with its ligands,whether CD14 acts as a co-receptor, the CpG sequence of the DNA ligand,and the cell type involved (Weber et al. 2012). The form ofadministration (local vs systemic) of CpG DNA can also determine whetherthe final outcome of TLR9 signalling is inflammatory or inhibitory(Wingender et al. 2006).

TLR9 is expressed in macrophages, B cells and dendritic cells, and theplasmacytoid dendritic cell (pDC) subset in particular. pDC arespecialised for the rapid production of the inflammatory cytokines knownas Type I interferons (IFN-I) following infection and act as centralregulators of inflammation and T cell responses as well as modulatingother innate immune cell functions (Takagi et al. 2011). The type 1interferons produced by pDC allow killing of intracellular infectionsand have potent effects on other immune cells, mediating DC maturation,monocyte and natural killer cell activation, enhancement of the adaptiveimmune response and ability to respond to other TLR stimuli. Thus, theimmunomodulatory functions of low dose naltrexone mediated throughantagonism of TLR9 would primarily affect pDC function, althoughdownstream effects following pDC modulation would be exerted on a widerange of cells.

The natural role of TLR9 is to stimulate an immune response. It may seemcounter-intuitive that TLR9 antagonism would be an effective therapy orsupportive care for a subject diagnosed with cancer, given that aneffective anti-tumour immune response, or agonism of the immune system,would be beneficial. However, TLR9 overactivity can have detrimentaleffects on the immune response; pDC activity is beneficial at theinitiation of an immune response, but chronic activation of TLR9 caninhibit T and B cell responses. Sustained TLR9 activation leads toimmunosuppression via indoleamine 2,3-dioxygenase (IDO), Programmed celldeath ligand 1 (PDL1) and TNF-related apoptosis-inducing ligand (TRAIL)(Boasso et al. 2011). In this instance IDO, which is produced by pDC inresponse to TLR7/9 signalling, exerts inhibitory effects, which areproposed to result in immunosuppression, and a lack of effective T cellresponses. TLR9 can also be tolerogenic to B cells in the presence ofapoptotic cells.

Although specialised for detection of microbial nucleic acids, TLR9 canrecognise, and be overstimulated by self-nucleic acids in certainpathological conditions. In systemic lupus erythematosus (SLE), self-DNAimmune complexes captured within neutrophil extracellular traps cansubsequently be recognised by TLR9 within pDC to trigger Type I IFNproduction; a similar situation can occur in psoriasis.

If TLR9 overactivity can result in immune dysregulation, it is thereforelogical that its antagonism by LDN would dampen down chronicinflammatory responses, in order to allow directed and beneficial immuneresponses to (re)emerge. Cancer, for example, is associated withinflammation and it has been suggested that about 25% of cancers arelinked to chronic inflammation (Mantovani 2011). Thus LDN treatment canreduce inflammation to enable the generation of effective anti-tumouradaptive immune responses, notably in cancers where inflammation isdriven by TLR9 overexpression in the cancer or microenvironment.

LDN-mediated inhibition of TLR9 could also allow beneficial immuneresponses to be triggered via other members of the TLR family; thiswould be of particular use in providing supportive care to a patientsuffering from any type of cancer. Approximately 89% of cases of cancerin the UK are diagnosed in people over 50 (Cancer Research UK),therefore the combination of cancer and age-related immune depression(immunosenescence) in a patient can result in substantial imbalance tothe immune system. Modulation of a subset of receptors such as TLR4 andTLR9 via naltrexone whilst allowing other members of the TLR family tobe stimulated, or stimulating the immune system via another pathway,could provide a simple inhibition of excess immune activity or allow amore subtle skewing of immune responses. Aside from treatment, thiscould provide improvements in quality of life to those suffering fromcancer and thus could be an effective strategy for supportive care.

As used herein “naltrexone” refers tomorphinan-6-one,17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxy-(5a)having the above chemical structure, and pharmaceutically acceptablesalts, solvates, hydrates, stereoisomers, clathrates and prodrugsthereof. The use of naloxone, a structural analogue of naltrexone whichthe skilled person would expect to also act as an antagonist of TLR9, iswithin the purview of the invention and is encompassed within the term“analogue” used in the description and the claims. Similarly, methylnaltrexone is also envisaged as a suitable analogue for use in allaspects of the invention. The preferred form of naltrexone is as itshydrochloride salt form.

As used herein, the terms “agonist” and “antagonist” have theirconventional meanings as used in the art.

As used herein, the term “overexpression of TLR9” refers to the level ofTLR9 mRNA and/or protein expressed in cells of a given tissue beingelevated in comparison to the levels of TLR9 as measured in normal cells(free from disease) of the same type of tissue, under analogousconditions. Said TLR9 mRNA and/or protein expression level may bedetermined by a number of techniques known in the art including, but notlimited to, quantitative RT-PCR, western blotting, immunohistochemistry,and suitable derivatives of the above.

As used herein, the term “overactivity of TLR9-mediated signalling”refers to the level of TLR9-mediated signalling activity, including theactivity of downstream effectors thereof, being elevated in comparisonto the level of such activity as measured in the same tissue type (freefrom disease), under analogous conditions. Said elevated activity ofdownstream effectors of TLR9-mediated signalling may not necessarily becaused by increased expression and/or activity of TLR9; a downstreameffector in an immune cascade/pathway mediated by inter alia, TLR9 mayexhibit overactivity due to aberrant activity of another upstreamcomponent of the cascade/pathway; however if the activity of saiddownstream effector is mediated (at least in part) by TLR9, thenantagonism of TLR9 may alter activity of said downstream effector fortherapeutic effect. Accordingly, disorders exhibiting overactivity ofdownstream effectors as described above may also be considered as beingcharacterised by overactivity of TLR9-mediated signalling. Downstreameffectors of TLR9-mediated signalling include, but are not limited to;cells, notably those associated with the immune system such as B cellsand dendritic cells (for example plasmacytoid dendritic cells) (saidcells may themselves express TLR9), wherein the activity of said cellsmay be mediated by inter alia, TLR9; and peptides/proteins, notablythose associated with an immune response such as cytokines (for exampleTNFa, IL-2, IL-6) and interferons (for example IFN-I), wherein theexpression, secretion and/or activity of said proteins may be mediatedby inter alia, TLR9. Downstream effectors of TLR9-mediated signallingmay be present in a different tissue to the TLR9 proteins which mediatetheir activity. Techniques known in the art which may be used todetermine the level of TLR9-mediated signalling activity include, butare not limited to; cell-based assays comprising analysis oftranscription and/or translation of a target gene and/or protein ofTLR9-mediated signalling; cell-based assays comprising analysis of cellphenotype and activity; cell-based assays comprising analysis ofphosphorylation, proteolytic processing and/or otherwise modification ofat least one downstream component of TLR9-mediated signalling; nucleicacid sequence analysis of TLR9 genes and/or RNA transcripts. Generally,but not necessarily, overexpression of TLR9 will result in overactivityof TLR9-mediated signalling. Overactivity of TLR9-mediated signallingmay not necessarily be the result of overexpression of TLR9, for examplein instances where the TLR9 gene and/or protein comprises activatingmutations.

As used herein, the terms “treating” and “treatment” and “to treat”refer to both 1) therapeutic measures that cure, slow down, and/or haltprogression of a diagnosed pathologic condition or disorder and 2)prophylactic or preventative measures that prevent and/or slow thedevelopment of a targeted pathologic condition or disorder. Thus, thosein need of treatment include those already with the disorder; thoseprone to have the disorder; and those in whom the disorder is to beprevented. In some instances, a subject is successfully “treated” forcancer according to the novel applications of the present invention ifthe patient shows one or more of the following: a reduction in thenumber of, or complete absence of, cancer cells; a reduction in thetumour size; inhibition of, or an absence of, cancer cell infiltrationinto peripheral organs including, for example, the spread of cancer intosoft tissue and bone; inhibition of, or an absence of, tumourmetastasis; inhibition of, or an absence of, tumour growth; reducedmorbidity and mortality; reduction in tumourigenicity, tumourigenicfrequency, or tumourigenic capacity of a tumour; reduction in the numberor frequency of cancer stem cells in a tumour; differentiation oftumourigenic cells to a non-tumourigenic state; or some combination ofeffects.

As used herein “supportive care”, in the context of the treatment of atumour/cancer, refers to that which helps the patient to cope withcancer and the treatment of it—from pre-diagnosis, through the processof diagnosis and treatment, to cure, continuing illness or death.Supportive care includes, but is not limited to, symptom control,complementary therapies, management of the side-effects of treatment,psychological support, rehabilitation, self help and support, palliativecare, end-of-life care. Accordingly, supportive care is not given withthe expectation of treating the tumour/cancer in question (in accordancewith the above definition), but improving quality of life for thepatient, for example through symptom relief.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth, proliferation and/orsurvival. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, myeloma and leukaemia.

As used herein, the terms “tumour” or “neoplasm” refers to any mass oftissue that results from excessive cell growth, proliferation and/orsurvival, either benign (noncancerous) or malignant (cancerous),including pre-cancerous lesions.

As used herein, the term “tumourigenic” refers to the functionalfeatures of a solid tumour stem cell including the properties ofself-renewal (giving rise to additional tumourigenic cancer stem cells)and proliferation to generate all other tumour cells (giving rise todifferentiated and thus non-tumourigenic tumour cells) that allow solidtumour stem cells to form a tumour. These properties of self-renewal andproliferation to generate all other tumour cells confer on cancer stemcells the ability to form palpable tumours upon serial transplantationinto an immunocompromised host (e.g., a mouse) compared tonon-tumourigenic tumour cells, which are unable to form tumours uponserial transplantation. It has been observed that non-tumourigenictumour cells may form a tumour upon primary transplantation into animmunocompromised host after obtaining the tumour cells from a solidtumour, but those non-tumourigenic tumour cells do not give rise to atumour upon serial transplantation.

As used herein, the term “tumour/cancer microenvironment” refers tonon-malignant cells, including extracellular features thereof, whichsupport the malignant cells of a tumour/cancer in their growth, survivaland/or metastasis. The non-malignant cells, also called stromal cells,may occupy or accumulate in the same cellular space as malignant cells,or the cellular space adjacent or proximal to malignant cells, whichmodulate tumour cell growth or survival. Non-malignant cells of thetumour microenvironment include, but are not limited to, fibroblasts,myofibroblasts, glial cells, epithelial cells, adipocytes, vascularcells (including blood and lymphatic vascular endothelial cells andpericytes), resident and/or recruited inflammatory and immune (e.g.,macrophages, dendritic cells, myeloid suppressor cells, granulocytes,lymphocytes, etc.), resident and/or recruited stem cells that arecapable of giving rise to or differentiating into any of the above-notednon-malignant cells, and any functionally distinct subtypes of theabove-noted cells as known in the art.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,canines, felines, rodents, and the like, which is to be the recipient ofa particular treatment. Typically, the terms “subject” and “patient” areused interchangeably herein in reference to a human subject.

As used herein, the term “immunomodulator” refers an agent whose actionon the immune system leads to an immediate or delayed enhancement orreduction of the activity of at least one pathway involved in an immuneresponse. Said response may be naturally occurring or artificiallytriggered; as part of the innate or adaptive immune system; or both.

In one embodiment, the pharmaceutical compositions of the presentinvention are used to modulate an immune response so as to treat asubject having a disorder characterised by TLR9 overexpression and/oroveractivity of TLR9-mediated signalling. The above features would beknown to the skilled person as characteristics of the disorder to betreated. Disorders characterised by TLR9 overexpression and/oroveractivity of TLR9-mediated signalling include, but are not limited toCrohn's disease, systemic lupus erythematosus, psoriasis, certaincancers. The subject to be treated may themselves be characterised ashaving TLR9 overexpression and/or overactivity of TLR9-mediatedsignalling. A subject may be characterised as such by way of adiagnostic test to determine the level of TLR9 expression and/orTLR9-mediated signalling (notably in cells pathologically associatedwith said disorder), relative to the levels in the same tissue type(when free from disease) under analogous conditions. Techniques known inthe art which may be used to determine the level of TLR9 expressionand/or TLR9-mediated signalling include, but are not limited to, thosestated above. Preferably, said disorder is a tumour/cancer. Morepreferably, the tumour/cancer and/or the microenvironment thereof ischaracterised as comprising TLR9 overexpression and/or overactivity ofTLR9-mediated signalling; such tumours/cancers include, but are notlimited to, those selected from the group consisting of breast cancer,cervical squamous cell carcinoma, gastric carcinoma, glioma,hepatocellular carcinoma, lung cancer, melanoma, prostate cancer,recurrent glioblastoma, recurrent non-Hodgkin lymophoma, colorectalcancer. However, the pharmaceutical compositions of the invention beingused in the treatment of subjects having other tumours/cancers, with theabove characteristics (the tumour/cancer and/or the microenvironmentthereof being characterised as comprising TLR9 overexpression and/oroveractivity of TLR9-mediated signalling) is also envisaged. The subjectmay also be administered, or has been administered, at least onechemotherapeutic agent in addition to naltrexone, preferably wherein theagent and dose thereof is selected so as to reduce immune suppression;such agents include, but are not limited to, Revlimid (preferablyadministered at a dose between 5 mg and 25 mg), Cyclophosphamide(preferably administered at a dose between 50 mg and 100 mg),Gemcitabine (preferably administered at an administered dose between 250mg/kg and 2000 mg) or carboplatin at a dose of 4-6 Areas Under the Curve(AUC).

In another embodiment, the present invention provides a method oftreating a subject having a disorder characterised by TLR9overexpression and/or overactivity of TLR9-mediated signalling; whereinthe method comprises administering to the subject, a pharmaceuticalcomposition of the invention. Said method of treatment has the sameoptional and preferred features as the use of the pharmaceuticalcomposition recited in the preceding paragraph.

In another embodiment, the pharmaceutical compositions of the presentinvention, optionally when administered with, prior to, or after atleast one other immunomodulator, are used to modulate an immune responsein order to provide supportive care to a subject having a tumour/cancer.Said tumours/cancers are not limited to those comprising TLR9overexpression and/or overactivity of TLR9-mediated signalling.Accordingly, examples include, but are not limited to carcinoma,lymphoma, blastoma, sarcoma, and leukaemia; more particular examples ofsuch tumours/cancers include squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, melanoma, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancers. If used, theimmunomodulator is preferably an immune agonist, i.e. an agent whichelicits and/or augments an immune response, for example a vaccine. Morepreferably, the immune agonist is selected from the group consisting ofcyclophosphamide; Revlimid; Imiquimod; a whole cell Mycobacteria,preferably a rough strain of Mycobacterium vaccae or Mycobacteriumobuense (as described in WO 07/071978); Daunorubicin; Oxaliplatin;5-Fluorouracil; Gemcitabine; Zometa. The subject to be given supportivecare may themselves be characterised as having TLR9 overexpressionand/or overactivity of TLR9-mediated signalling. A subject may becharacterised as such by way of a diagnostic test to determine the levelof TLR9 expression and/or TLR9-mediated signalling, relative to thelevels in the same tissue type (when free from disease) under analogousconditions. Techniques known in the art which may be used to determinethe level of TLR9 expression and/or TLR9-mediated signalling include,but are not limited to, those stated above. Wherein the subject ischaracterised as such; preferably the cells of the immune system inwhich TLR9 is natively expressed are characterised as havingoverexpression of TLR9. Such cells include, but are not limited tomacrophages; B cells; and dendritic cells, notably plasmacytoiddendritic cells. Also wherein the subject is characterised as such; thetumour/cancer, and or the microenvironment thereof, may be identified ashaving TLR9 overexpression and/or overactivity of TLR9-mediatedsignalling.

In another embodiment, the present invention provides a method ofproviding supportive care to a subject having a tumour cancer. Saidmethod of providing supportive care has the same optional and preferredfeatures as the use of the pharmaceutical composition recited in thepreceding paragraph.

According to the embodiments outlined above, naltrexone, as part of thepharmaceutical composition, is preferably administered to a subject at adose between 0.01 mg/kg and 0.08 mg/kg, more preferably between 0.03mg/kg and 0.06 mg/kg, most preferably between 0.04 mg/kg and 0.05 mg/kg.The composition can be administered in any conventional way.Administration can be by oral or parenteral administration, preferablyoral administration. However, other routes of administration are alsoenvisaged.

It has also been realised that naltrexone preferably low dose naltrexone(including its pharmaceutically acceptable salts, solvates, hydrates,stereoisomers, clathrates, prodrugs and analogues thereof) are suitablefor use as a vaccine adjuvant. The naltrexone may be administeredsimultaneously, separately or sequentially with an immunogen. Forexamples the naltrexone may be administered either before or afteradministration of the immunogen (vaccine). In this aspect, thenaltrexone is believed to stimulate cytotoxic T cells activation via thestimulation of dendritic cells. Accordingly, naltrexone can therefore beused in this context to help promote an immune response against variousdisorders, including cancer, e.g. metastatic melanoma.

There is therefore envisaged vaccine compositions comprising naltrexoneand an immunogen.

The invention is now illustrated by the following non-limiting Examples.

EXAMPLES Example 1—Identification of Naltrexone as an Antagonist of TLR9

Low dose naltrexone (LDN) was tested in agonist and antagonist assays ona panel of TLR and NOD transfectants (TLR2, TLR3, TLR4, TLR5, TLR7,TLR8, TLR9, NOD1, NOD2). Screening assays were performed byCayla-lnvivogen (Toulouse, France). In agonist assays, LDN did notstimulate signalling through any of the receptors tested. In antagonistassays, the mild antagonist effect observed by Hutchinson et al. (2008)for TLR4 was replicated, but a more striking effect was seen for TLR9,for which LDN inhibited recognition of CpG DNA (FIG. 1). A subsequenttitration suggested an IC50 of approximately 50 μM (FIG. 2).

Example 2—LDN in the Treatment of Melanoma

Patient with chemotherapy-resistant metastatic melanoma showed a markedclinical response to LDN in combination with vitamin D3, which wasmaintained for nine months.

The patient had a recurrent head and neck melanoma and had previouslybeen treated with a vaccine and radiotherapy, but had failed to respondto this and had developed a new metastatic disease in the chest.

Low dose naltrexone was administered as a supportive treatment prior tomore radiotherapy. Within 48 hours the patient developed marked vitiligowhich is a sign of activated effective cytotoxic T cells against theantigens associated with melanoma. It was concluded that the naltrexonehad activated the vaccine. The patient responded to radiotherapy to thechest lesions.

Example 3—LDN in the Treatment of Prostate Cancer

Patient with hormone-refractory prostate cancer and markedlymphadenopathy being treated with Mycobacterium vaccae (aTLR2-stimulating vaccine) showed a marked response with the addition ofLDN, including a drop in prostate-specific antigen levels.

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1. A pharmaceutical composition comprising naltrexone or an analoguethereof, for use in the treatment of a subject having a disorder whichis characterised by TLR9 overexpression and/or overactivity ofTLR9-mediated signalling.
 2. A pharmaceutical composition according toclaim 1, wherein the subject is characterised as having TLR9overexpression and/or overactivity of TLR9-mediated signalling.
 3. Apharmaceutical composition according to claim 1, wherein the disorder isa tumour/cancer.
 4. A pharmaceutical composition according to claim 3,wherein the tumour/cancer is selected from the group consisting ofbreast cancer, cervical squamous cell carcinoma, gastric carcinoma,glioma, hepatocellular carcinoma, lung cancer, melanoma, prostatecancer, recurrent glioblastoma, recurrent non-Hodgkin lymphoma,colorectal cancer.
 5. A pharmaceutical composition according to claim 1,wherein the subject is or has been administered at least onechemotherapeutic agent selected from the group consisting of Revlimid,cyclophosphamide, Gemcitabine and carboplatin.
 6. A pharmaceuticalcomposition according to claim 5, wherein Revlimid is administered at adose between 5 mg and 25 mg, cyclophosphamide is administered at a dosebetween 50 mg and 100 mg, and/or Gemcitabine is administered at a dosebetween 250 mg and 2000 mg.
 7. A pharmaceutical composition comprisingnaltrexone or an analogue thereof, for use in the supportive care of asubject having a tumour/cancer.
 8. A pharmaceutical compositionaccording to claim 7, wherein the subject is characterised as havingTLR9 overexpression and/or overactivity of TLR9-mediated signalling. 9.A pharmaceutical composition according to claim 8, wherein macrophages;B cells; and/or dendritic cells, preferably plasmacytoid dendriticcells; of the subject are characterised as having TLR9 overexpression.10. A pharmaceutical composition according to claim 7, wherein saidsubject is or has been administered at least one other immunomodulator,preferably an immune agonist.
 11. A pharmaceutical composition accordingto claim 10, wherein the immune agonist is selected from the groupconsisting of cyclophosphamide, Revlimid, Imiquimod, a whole cellMycobacteria, Daunorubicin, Oxaliplatin, 5-Fluorouracil, Gemcitabine,Zometa.
 12. A pharmaceutical composition according to claim 11, whereinthe whole cell Mycobacteria is a rough strain of Mycobacteria obuense orMycobacteria vaccae.
 13. A pharmaceutical composition according to claim1, wherein naltrexone, as part of said composition, is to beadministered to the subject at a dose between 0.01 mg/kg and 0.08 mg/kg,preferably between 0.03 mg/kg and 0.06 mg/kg, more preferably between0.04 mg/kg and 0.05 mg/kg.
 14. A method of treating a subject having adisorder which is characterised by TLR9 overexpression and/oroveractivity of TLR9-mediated signalling; wherein the method comprisesadministering to the subject, a pharmaceutical composition comprisingnaltrexone or an analogue thereof.
 15. A method according to claim 14,wherein the subject is characterised as having TLR9 overexpressionand/or overactivity of TLR9-mediated signalling.
 16. A method accordingto claim 15, wherein the method additionally comprises testing thesubject for TLR9 overexpression and/or overactivity of TLR9-mediatedsignalling.
 17. A method according to claim 14, wherein the disorder isa tumour/cancer.
 18. A method according to claim 17, wherein thetumour/cancer is selected from the group consisting of breast cancer,cervical squamous cell carcinoma, gastric carcinoma, glioma,hepatocellular carcinoma, lung cancer, melanoma, prostate cancer,recurrent glioblastoma, recurrent non-Hodgkin lymophoma, colorectalcancer.
 19. A method according to claim 14, wherein the subject is orhas been administered at least one chemotherapeutic agent selected fromthe group consisting of Revilimid, cyclophosphamide, Gemcitabine andcarboplatin.
 20. A method according to claim 19, wherein Revlimid isadministered at a dose between 5 mg and 25 mg, cyclophosphamide isadministered at a dose between 50 mg and 100 mg, and/or Gemcitabine isadministered at a dose between 250 mg and 2000 mg.
 21. A method ofproviding supportive care to a subject having a tumour/cancer,comprising administering to the subject, a pharmaceutical compositioncomprising naltrexone or an analogue thereof.
 22. A method according toclaim 21, wherein the subject is characterised as having TLR9overexpression and/or overactivity of TLR9-mediated signalling.
 23. Amethod according to claim 22, wherein macrophages; B cells; and/ordendritic cells, preferably plasmacytoid dendritic cells; of the subjectare characterised as having TLR9 overexpression.
 24. A method accordingto claim 21, wherein said subject is or has been administered at leastone other immunomodulator, preferably an immune agonist.
 25. A methodaccording to claim 24, wherein the immune agonist is selected from thegroup consisting of cyclophosphamide, Revlimid, Imiquimod, a whole cellMycobacteria, Daunorubicin, Oxaliplatin, 5-Fluorouracil, Gemcitabine,Zometa.
 26. A method according to claim 25, wherein the whole cellMycobacteria is a rough strain of Mycobacteria obuense or Mycobacteriavaccae.
 27. A method according to claim 14, wherein naltrexone, as partof said composition, is to be administered to the subject at a dosebetween 0.01 mg/kg and 0.08 mg/kg, preferably between 0.03 mg/kg and0.06 mg/kg, more preferably between 0.04 mg/kg and 0.05 mg/kg. 28-35.(canceled)